SUMMARY The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair; control of this process is therefore of fundamental importance in both muscle development and muscle regeneration after injury. The tendency for myogenic cells cultured densely to differentiate and, conversely, the resistance to differentiation of cells at low density has been called the 'Community Effect'; understanding this phenomenon represents a basic question in muscle biology. Based on our initial observation that EphA7, a juxtacrine signaling molecule, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo we examined the muscle phenotype of EphA7-/- mice. We found that their hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exposure to exogenous EphA7 extracellular domain will rescue the null phenotype, and will also accelerate commitment to differentiation in WT cells, which led us to propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation via reverse signaling. The experiments we propose in Aims 1 and 2 will address the role of EphA7 in myogenic commitment in both the myocyte (How does commitment to differentiation lead to expression of EphA7?) and the myoblast (How does receiving an EphA7 signal lead to commitment to differentiation?). Once they have differentiated, myocytes must fuse with each other or with a growing myotube in order to generate a functional muscle cell (the contractile myocyte fiber), thus this also represents a critical process in both development and regeneration. However, the molecular requirements for fusion in mammalian muscle cells have been elusive. Our data suggest EphA7 also promotes myogenic fusion, possibly via different molecular mechanisms/interactions from its role in promoting myogenic differentiation. The experiments we propose in Aim 3 will test the ability of EphA7 to promote fusion in myogenic and nonmyogenic cells, determine whether it associates with the cell-surface fusogen myomaker, and identify other protein-protein interactions it is participating in at the interface of myocytes and growing myotubes in cis (on the same cell membrane) or in trans (on opposing cell membranes). Collectively, these studies will address the molecular mechanisms regulating two fundamental cellular processes during myogenic differentiation; they also have the potential to provide insight into potential innovations in muscle stem cell expansion in vitro and thus applications in tissue engineering and regenerative medicine.